The incidence of pancreatic cancer, colon cancer, liver cancer, melanoma and cervical cancer in the worldwide population is increasing. Effective treatments for these diseases are urgently needed. In many types of cancer including leukemia, melanoma, pancreatic, colon, renal cell carcinoma, lung, prostate, breast, brain, cervical and liver cancers, the cancer cells are auxotrophic for arginine since they lack of expression of argininosuccinate synthetase (ASS), making these cancers excellent targets for arginine depletion therapy.
Arginine is a semi-essential amino acid for humans and other mammals. It can be synthesized from citrulline via a two step process catalyzed by the urea cycle enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL). Arginine can be metabolized to ornithine by the enzyme arginase, and ornithine can be converted to citrulline by ornithine carbamoyltransferase (OTC) in the mitochondria. The citrulline can be utilized to synthesize arginine again. Normal cells usually do not require an exogenous supply of arginine for growth because of the abundant catalytic activity of ASS and ASL. In contrast, many types of cancers do not express ASS and therefore are auxotrophic for arginine. Their growth is dependent on arginine solely obtained from blood circulation. Therefore, targeting circulating arginine by using arginine-degrading enzymes is a feasible strategy to inhibit ASS-negative tumor growth [Feun et al., Curr. Pharm. Des. 14:1049-1057 (2008); Kuo et al., Oncotarget. 1:246-251 (2010)].
Arginine can be degraded by arginase, arginine decarboxylase, and arginine deiminase (ADI). Among them, arginine deiminase (ADI) appears to have the highest affinity for arginine (a low Km value). ADI converts arginine to citrulline and ammonia, the metabolites of the urea cycle. Unfortunately, ADI can only be found in prokaryotes e.g. Mycoplasma sp. There are some problems associated with the isolation and purification of ADI from prokaryotes. ADI isolated from Pseudomonas putida fails to exhibit efficacy in vivo because of its low enzymatic activity in neutral pH. ADI produced from Escherichia coli is enzymatically inactive and subsequently requires multiple denaturation and renaturation process which raises the subsequent cost of production.
As the native ADI is found in microorganisms, it is antigenic and rapidly cleared from circulation in a patient. The native form of ADI is immunogenic upon injection into human circulation with a short half-life (˜4 hours) and elicits neutralizing antibodies [Ensor et al., Cancer Res. 62:5443-5450 (2002); Izzo et al., J. Clin. Oncol. 22:1815-1822 (2004)]. These shortcomings can be remedied by pegylation. Among various forms of pegylated ADI, ADI bound with PEG (molecular weight 20,000) via succinimidyl succinate (ADI-PEG 20) has been found to be an efficacious formulation. However, the activity of ADI after pegylation is greatly decreased on the order of 50% [Ensor et al., Cancer Res. 62:5443-5450 (2002)]. The previous attempts to create pegylated ADI resulted in materials that are not homogenous (due to the random attachment of PEG on protein surface Lys residues) and also difficult to characterize and perform quality control during the manufacturing process. Also, PEG is very expensive, greatly increasing the production cost. After the intravenous injection of pegylated ADI in vivo, leakage or detachment of free PEG is observed and the ADI (without PEG) can elicit the immunogenicity problem. Therefore, there is a need for improved cancer-treatment compositions, particularly, improved cancer-treatment compositions that have enhanced activity and in vivo half-life.